Method of treating atherosclerosis using 4-hydroxymethyl-1-keto-1,2-dihydrophthalazine or acid salts thereof

ABSTRACT

OR A PHARMACEUTICALLY ACCEPTABLE ACID SALT THEREOF IN A DOSE OF 1 TO 100 MG./KG/ BODY WEIGHT/DAY.   1-(O=),4-(HO-CH2-)-1,2-DIHYDROPHTHALAZINE   1. A METHOD OF TREATING ATHEROSCLEROSIS WHICH COMPRISES ADMINISTERING TO A PATIENT SUFFERING FROM ATHEROSCELEROSIS THE COMPOUND OF THE FOLLOWING FORMULA

United States Patent Cffice Patented Oct. 8, 197 4 3,840,662 METHOD OF TREATING ATHEROSCLEROSIS USING 4-HYDROXYMETHYL-1-KETO-1,2-DI- HYDROPHTHALAZINE R ACID SALTS THEREOF Michiro Inoue, 26-3, 6-chome, Kokuryo-cho, Chofu-shi; Masaynki Ishikawa, 14-13, 3-chome, Akazutsumi, Setagaya-ku; Takashi Tsuchiya, 13 Kitamachi, Shinjuku-ku; and Takio Shimamoto, 17-25, S-chome, Minamikoiwa, Edogawa-ku, all of Tokyo, Japan No Drawing. Filed July 24, 1972, Ser. No. 274,512 Int. Cl. A61k 27/00 US. Cl. 424-250 3 Claims ABSTRACT OF THE DISCLOSURE Pharmaceutical composition comprising an effective amount of a compound of the following formula CIH2OH or a pharmaceutically acceptable acid salt thereof, and diluent; method of treating hemorrhage, thrombosis and atherosclerosis by using said composition; and process for production of a compound of said formula.

This invention relates to a novel process for producing known 4-hydroxymethyl-l-keto-l,Z-dihydrophthalazine, a process for producing acid salts thereof, a pharmaceutical composition containing said dihydrophthalazine or pharmaceutically acceptable acid salts thereof, and to a method of treating hemorrhage, thrombosis or atherosclerosis.

The 4 hydroxymethyl-l-keto 1,2 dihydrophthalazine expressed by the following formua (I) is a known compound having a melting point of 204 C. [Annual Report of Department of Pharmacy, Kanazawa University, Japan, Vol. 12, l-6 (1961)]. It has not however, been known at all that this compound has pharmaceutical effects. The above-mentioned report states that the compound of formula (I) was obtained by brominating o-acetylbenzoic acid, hydrolyzing the product with an acid, and reacting the hydrolyzate with hydrazine, in accordance with the method disclosed in Ber. 40, 72 1907).

This report does not disclose anything about the pharmaceutical effects of the compound obtained nor its possible utility. Furthermore, it states that the compound of formula (I) was obtained in a very low yield of several percent to 10% by this disadvantageous operation requiring many steps.

It has now been found by the inventors of this specification that the compound of formula (I) is a substance having pharmaceutical effects, especially for the treatment (including the prevention and curing) of hemorrhage, thrombosis and atherosclerosis and also having low toxicity (for example, LD more than 4,500 mg./kg. mouse).

Further study has led to the discovery that the compound of formula (I) can be easily produced in a high yield above by simply reacting a compound of forwherein R is an alkoxy group having 1 to 5 carbon atoms or a halogen atom, with an alkali metal borohydride in a solvent, and that the compound of formula (I) forms unkown addition salts with organic or inorganic acids, which addition salts exhibit pharmaceutical effects, which are the same as, or superior to, those of th compound of formula (I).

It is known that generally lithium aluminum hydride is used in order to obtain a primary alcohol by reducing a carboxylic acid ester, and the reaction does not proceed essentially with sodium borohydride [Angew. Chem., 73, 81 (1961); H. C. Brown Hydroboration 242 (1963)]. It is also well known that when a notrogencontaining heterocyclic compound, especially a diazine compound such as phthalazine or quinoxaline is treated with lithium aluminum hydride, the nitrogen-containing hetero ring is easily reduced [Jikken Kagaku Koza (or Lectures on Experimental Chemistry), Vol. 17, page 61].

Surprisingly, we have found contrary to the expectation from the above-mentioned prior knowledge that by the reaction of the compound of formula (II) with an alkali metal borohydride, the compound of formula (I) can be formed in high yield.

According to the present invention, therefore, the compound of formula (I) can be obtained in high yield from an inexpensive material by a simple reaction operation in which the reaction steps are very much shortened.

Accordingly, a primary object of this invention is to provide a novel pharmaceutical composition which is useful especially for treating hemorrhage, thrombosis and atherosclerosis.

A second object of this invention is to provide an advantageous process for producing the compound of formula (I) having the above-mentioned pharmaceutical effects.

A third object of this invention is to provide a method of treating hemorrhage, thrombosis and atherosclerosis utilizing the pharmaceutical efiects that have been newly discovered.

Many other objects of this invention along with its advantages will become more apparent from the following description.

The compounds of formula (11) used in this invention can be easily obtained by a known method. For example, it can be easily produced, in accordance with the method disclosed at page 1316, Vol. 68, Journal of The American Chemical Society, by oxidizing naphthalene with potassium permanganate, reacting the oxidized product with hydrazine, and esterifying the resultant 4-carboxy-l-keto- 1,2-dihydrophthalazine in a customary manner or treating it with thionyl chloride to convert it to the acid chloride.

Examples of compounds according to formula (H) include 4-methoxycarbonyll-keto-l,Z-dihydrophthalazine, 4-ethoxycarbonyl-l-keto-1,2-dihydrophthalazine, 4-propoxycarbonyl-l-keto-1,2-dihydrophthalazine, 4-butoxycarbonyl-l-keto-1,2-dihydrophthalazine, 4-amyloxycarbonyl-l-keto-1,2-dihydrophthalazine, 4-chlorocarbonyl-1-keto-1,2-dihydrophthalazine, and 4-bromocarbonyl-1-keto-1,2-dihydrophthalazine.

3 As the alkali metal borohydride, there can be used, for example, lithium borohydride, sodium borohydrlde, potassium borohydride, or sodium trimethoxy borohydridef In the production of the compound of formula (I), the alkali metal borohydride is used in an amount of,

for example, 0.8 to 20 mols, preferably 1 to 3 mols per mol of the compound of formula (II), and the reaction is carried out in a solvent. It is advantageous to carry out the reaction in the presence of a metal halogenide, such as calcium chloride, magnesium bromide, lithium chloride, lithium bromide or lithium iodide. When R in formula (II) is an alkoxy group, water or a C -C aliphatic alcohol such as methanol, ethanol, propanol or amyl alcohol can be used as the solvent. The use of C -C alcohols is preferred.

When R is a halogen atom in formula (II), ordinary inert solvents such as benzene, ethyl ether, dioxane or tetrahydrofuran or mixtures thereof can be used as the solvent. When R is a halogen atom, it is sufficient that the alkali metal borohydride is used in an amount of about 1 to 5 mols per mol of the phthalazine derivative. The reaction can be carried out usually at 0 to 250 C., preferably 0 to 200 C., more preferably 0 to 100 C. When R in formula (II) is an alkoxy group, it is preferred that the reaction be carried out at 0 to 100 C. Usually, the reaction ends in 0.5 to 3 hours, but the reaction may also be carried out at low temperatures for long periods of time. The product can be easily isolated, and purified.

According to this invention, there are provided pharmaceutical compositions comprising an effective amount of the compound of the formula CHiOH or a pharmaceutically acceptable acid addition salt thereof, and a diluent.

This composition has outstanding pharmaceutical effects especially for the treatment of hemorrhage, thrombosis and atherosclerosis. Such thrombosis include, for eX- ample, cerebral thrombosis, coronary thrombosis and peripheral thrombosis. Examples of the atherosclerosis include cerebral atherosclerosis, coronary atherosclerosis, arteriosclerosis, oblitrance, thromboangitis oblitrance, thromphlebitis, angiopathy of diabetes mellitus, or nephropathy of diabetes mellitus.

The effective amount of the compound of formula (I) can be freely changed according to a particular dose intended, but usually is about 0.1 to 80% based on the combined amount of the diluent and the compound of formula (I). In short, it may be any desired concentration required for administration in a dose of 1 to 100 mg./kg. body weight/day.

The diluent may be a liquid or solid, and the term diluent is used in this invention to denote also adjuvants. Examples of the liquid diluent include distilled water for injection, isotonic sodium chloride solution, Ringers solution, Lockes solution, polyethylene glycol, ethyl alcohol, propylene glycol, glycerol, liquid paratlin, and vegetable oil. The solid diluent includes, for example, sodium chloride, glucose, polyvinyl pyrrolidone, lactose, starch, methyl cellulose, sucrose, polyethylene glycol, white vaseline, cetyl alcohol, cacao butter and spermaceti.

The composition of this invention can be used in various forms such as injection, infusion, powder, tablet, granule, capsule, troche, solution, elixir, suspension, spirit, syrup, limonad, ointment, eye drop and suppository.

The composition of this invention can be administered by various means, for example, parenteral administration such as injection including intravenous injection, intraarterial injection, intracutaneous injection and infusion, coating, eye dropping and insertion, and oral administration.

The invention will now be described further by the following Examples.

EXAMPLE 1 To a solution of NaBH in ethanol which was prepared by dissolving 19 g. NaBH, in 120 ml. ethanol at 010 C., was added 12 g. 4-ethoxycarbonyl-1-keto-1,2-dihydrophthalazine 12 g. at 0-10 C. with stirring. To the suspension so obtained, was added a solution of 3 g. calcium chloride in 36 ml. ethanol, and then the reaction' mixture was stirred further for 5 hours at room temperature. The ethanol was distilled off under reduced pressure, and the residue was dissolved in water. The solution was adjusted to pH 6-7 with acetic acid and dried under reduced pressure. The residue was extracted continuously with chloroform using a Soxlet-apparatus. The chloroform extract was distilled off, and the residue was recrystallized from methanol to yield 4-hydroxymethyl-l-keto-1,2-dihydrophthalazine melting at 206208 C. The yield of the product was 8.6 g. (90%).

UV spectrum:

225 m;L(e-'=16,100), 245 m,u(e=7,700), 253 m,u.(e=8,200), 281 m,u.(e=6,400), 300 In,u.(e=4,500), 312 m;(s=2,800).

EXAMPLE 2 A mixture of 3 g. 4-carboxy-l-keto-1,2-dihydrophthalazine 3 g. and 40 ml. thionyl chloride was refluxed gently at 7080 C. for one hour on a water bath. The excess of thionyl chloride was then distilled off, and the residue was dissolved in 30 ml. dioxane. To the dioxane solution, was added portionwise 2 g. sodium borohydride at 0- 10C. with stirring. The reaction mixture was stirred further for one hour, and then the solvent was distilled off. The residue was recrystallized from water to yield 4-hydroxymetliyl-1-keto 1,2 dihydrophthalazine 1.2 g. melting at 201 202 C.

EXAMPLE 3 Examples of pharmaceutical formulations are as follows:

Injection and infusion:

Compound B 1 g., sodium chloride 0.9 g., and distilled water for injection 100 ml.

Compound B 1 g., glucose 5 g., and distilled water for injection 100 ml.

Compound B 1 g., polyvinylpyrroridone 10 g., and distilled water for injection 90 ml.

Compound B 1 g., propyleneglycol 20 ml., and distilled water for injection ml. lPowder:

Compound A 1 g. and lactose l g.;

Compound A 10 g. and lactose g.;

Compound A 1 g. and lactose 99 g.;

Compound A 1 g. and starch 99 g.;

Compound A 1 g. and lactose or starch or their mixture 98.99 and yellow dye 0.01 g. Tablet:

Compound A 50 mg., lactose 100 g., starch 43 mg., methyl cellulose 5 mg., and magnesium stearate 2 mg. Granular:

Compound A 25%, lactose 50%, starch 22.5% and methyl cellulose 2.5%. Capsule:

Compound A 50 mg., glucose 100 mg., starch 4s mg,

magnesium stearate 1 mg., and talc 1 mg;

Compound A 50 mg., and vegetable oil 50 mg.

Troche:

Compound A 50 mg., sucrose 898 mg., magnesium stearate 50 mg., and yellow dye 2 mg. Solution:

Compound B and purified water 90%.

Elixir:

Compound B 10 g., ethanol 10 ml., glycerin 20 ml.,

sucrose 35 g., and purified water 30 ml.

Suspension:

Compound A 10 g., sodium methyl cellulose carboxylate 1 g., yellow dye 0.02 and purified water 90 ml.

Spirit:

Compound A 10%, purified water 10% and ethanol Syrup:

Compound B 10 g., sucrose 50 g., ethanol 2 ml., and purified water 40 ml. Limonad:

Compound B 10 g., citric acid 0.3 g. sucrose 5 g., and

purified water 85 ml.

Ointment:

Compound A 1%, white vaseline 94%, and liquid paraf- Compound A 1 g., white Vaseline 39 g., cetyl alcohol 18 g., sesquioleic acid sorbitate 5 g., lauromagrogol 0.5 g., boric acid 0.2 g., and purified water 40 ml.

Compound A 1 g., polyethyleneglycol-400 54 g., polyethyleneglycol-4000 45 g.

[Eye drop:

Compound B 1 g., isotonic agent (boric acid or sodium chloride) suitable amount, and distilled water 100 ml. Suppository:

Compound A 0.1 g., cacao butter 1.6 g., spermaceti Compound A 0.1 g., polyethyleneglycol-4000 0.3 g., and

polyethylene-glycol-15000 1.6 g.

(Compound A and Compound B in the above formulation mean 4-hydroxymethyl-l-keto-l,2-d ihydrophthalazine and 4-hydroxymethyl-1-keto-l,2-dihydrophthalazine hydrochloride, respectively.)

EXAMPLE 4 Eifect of 4-hydroxymethyl-l-keto-1,2-dihydrophthalazine for the prevention of experimental atherosclerosis weeks. Thereafter 5 rabbits from the above treated groupwere sacrificed. The remaining 90 rabbits were divided into six groups. While all of the groups were fed with standardized food, five groups were each administered with 10 mg. per kg. of 4-hydroxymethyl-1-keto-1,2-dihydrophthalazine, the remaining one group with placebo consisting of potato starch. After 6, l0 and 20 weeks, each 5 rabbits of the six groups were sacrificed, and the total contents of cholesterol in the arterial wall were es timated by gas chromatography. The results are shown below in Table 1.

1. The total-cholesterol content at the end of the first 15 weeks was 40.6-12.1 gjmg. dry Weight.

2. The total-cholesterol contents of the six groups are indicated in the following table. The contents are shown in g. unit per one mg. of the dried arterial wall. The values shown below are the average one of each 5 rabbits.

TABLE 1 Period Cholesterol Group administered with- (week) content 13.)

Placeb 6 29. 3=!=1. 6 10 31.7:h2.5 20 24. 91:28. 1

Do 6 30.6=|=2.4 10 21. 25:2.3 20 23. 8=t=6.2

2,6-bis(hydroxymethyl)pyrldine-di-N- methylcarbamate(pyrldlnolcarbamate). 8 26. 05:5. 6 10 17. 8:1:2. 3 20 10. 6:1:24

Pyrldlnolcarbamate 6 27. 312. 6 10 16. 0&2. 6 20 9. 83:14

4-h (1 th I! 2-dlh dro hthalszine 6 28.1i3.4 y roxyme y y p 10 ,13.2i2. 1 20 6.3i2.5

Do 6 26. 5=t;4.0 53 12. 25:21

EXAMPLE 5 4-hydroxymethyl-l-keto-1,2-dihydrophthalazine showed a preventive effect for the enhancement of coagulability and thrombogenicity by one shot treatment of animal with cholesterol or adrenaline.

After oral administration of 10 rngJkg. of the compound of the present invention to a rabbit, intensity of platelet aggregation induced by adenisine diphosphate was measured by Borns method (Born, J. Physiol. 162, 67 (1962), see also OBrien, 1. Clin. Path. 15, 452 (1962), Lancet, 1, 779 (1968).

A rabbit was injected with adrenaline (1 gjkg.) 3 hours after oral administration of the sample. Five minutes after the injection, 4.5 ml. of blood was taken from the carotid artery and then diluted with 0.5 ml. of 3.8% solution of sodium citrate. After centrifugation of the blood at 1000 g. for 30 minutes, aliquots each of 0.9 ml. were taken from the supernatant. To the aliquots were added each 0.1 ml. of 3 l0- molar and 10- molar solutions of adenosine diphosphate. Therefore the molar concentrations of ADP in the serum were 3 10- and 10- respectively. The intensity of platelet aggregation was measured using a platelet aggregation meter (Model 169, Evans Elect. Ltd. England). Intensities of the ADP- induced platelet aggregation were shown as percentage of the pre-injection value. As shown in Table 2, the compound of this invention showed lower values of the ADP- induced platelet aggregation, thus preventing enhancement of coagulability and thrombogenicity.

The etfcct of the compound of the present invention on the clinical course and relapse rate of patients after their apopletic attack of cerebral hemorrhage or cerebral thrombosis was estimated in a controlled study.

Fifty-six patients admitted within 30 days after the stroke were subjected to the study. They were uniformly divided into A group (a control group) and B group (a group treated with the compound of the present invention) at their admission and also matched to sex, age, symptoms and so on. As shown in Table 3, there were 27 patients in the A group and 29 patients in the B group:

there were 9 patients with hemorrhages and 18 with thrombosis in the A group and 9 were patients with hemorrhages and 20 with thrombosis in the B group. There were no significant dilferences between the two groups with respect to sex, age, symptoms, complications, early history, onset of observation and period of observation. The diagnosis was made mainly according to the criterion of Dr. Milikan et al.

One gram of the compound of the present invention was administered orally to the B group daily. All other medical treatment was identical in the two groups. The clinical effect of the compound on the disturbance of consciousness, speech and motor function was studied. The degree of impairment of each symptom was assessed in each patient at admission and days later. Five assessment grades were used ranging from no disability to totally disabled. A distinct improvement of one grade or even an improvement in the same grade in any one of three symptoms during th is 10 day period was defined as clinical improvement.

TABLE 3 Group A Group B Number of cases 27 29.

Hemorrhage 9 9. Thombosis- IR 20.

Sex:

Male- 21 23. Female 6 6. Age- 38-86 years 37-86 years. SymBtom:

isturbance of consciousness (eima).- 10 (2) 13 (1). Speech, (aphasis) 21 3 22 Motor function (hemlplegla) 27 (10) 29 (9). Complications:

' Hypertension. 17 15.

Diabetes memh" 1. 2. Hypercholesterolemia 7 5. Abnormal ECG 9 11.

Early history:

Apoplexy 4 3. Arteriosclerotic 7 5. Hypertension- 11.

I Onset. of observation 8 7 1 6 days- 9 7 1 6 ays. Period of observation 8 days-6 8 daysfi months. months.

In the A group, 15 of 27'cases or 55.6% showed clinical improvement. On the other hand, in the B group clinical improvement was observed in 25 of 29 cases or 86.2%. This dilference is statistically significant at the 5% level.

What is claimed is:

1. A method of treating atherosclerosis which comprises administering to a patient suffering from atherosclerosis the compound of the following formula REFERENCES CITED Chemical Abstracts, vol. 59, formula index, 1963, p. 183 F.

JEROME D. GOLDBERG, Primary Examiner imrso STATES PATENT oFFi'CE CERTIFEATE OF CORRECTION Patent No. 3,840,662 Dated October 8, 1974 i Invenwfls v Michiro INOUE ET AL It is certified that error appears in the above-identified patent and that said Letters Patent are hereby corrected as shown below:

In the Heading, insert patentees Foreign Application Priority data as follows:

-- Claims priority, application Japan, August'S, l97l,

Signed and sealed this 18th day of February 1975.

(SEAL) p t: Attes C. MARSHALL DANN RUTH C. MASON Commissioner of Patents Attesting Officer and Trademarks 1 FORM PC4050 (1069) 

1. A METHOD OF TREATING ATHEROSCLEROSIS WHICH COMPRISES ADMINISTERING TO A PATIENT SUFFERING FROM ATHEROSCELEROSIS THE COMPOUND OF THE FOLLOWING FORMULA 